Recombinant poxvirus not having a functional 3beta-hsd gene

ABSTRACT

A recombinant  poxvirus , wherein the  poxvirus  genome does not comprise a functional gene encoding a 3β-hydroxysteroid dehydrogenase/Δ 5 -Δ 4 isomerase, may be used for vaccination against infection by a poxvirus. The recombinant  poxvirus  in which, additionally, the  poxvirus  genome comprises a non-poxvirus gene or a fragment of a non-poxvirus gene which gene or fragment encodes an antigen, for example, of a pathogenic agent, may be used to induce an immune e response against the antigen, for example, for vaccination against infection by the pathogenic agent.

The present invention relates to poxviruses with improved properties foruse as a vaccine and to methods associated therewith.

BACKGROUND

In the mid to late 20^(th) century, vaccinia virus (VV) was used tovaccinate large numbers of humans against variola virus, the causativeagent of smallpox. The vaccination campaign was highly successful andsmallpox was declared as eradicated by WHO in 1980. The success of VV asa live vaccine was due, in part, to its low production cost and theability to administer the vaccine by simple dermal abrasion. Thoseadvantages make VV and other poxviruses attractive for use as the basisfor recombinant viral vaccines and, consequently, VV is currently themost explored recombinant viral vaccine (see, for example, Smith et al.,1983, Panicali et al., 1983, Moss et al., 1996, and Dorrell et al.,2001).

The genome of VV strain Copenhagen has been sequenced (Goebel et al.,1990) and it was found to comprise in the region of 200 genes. Of thosegenes, at least one third are dispensible for virus replication in vitro(Perkus et al., 1991). VV possesses several genes which have beenproposed to aid evasion or suppression of the host immune system.

A VV open reading frame (ORF) denoted as SalF7L in Western Reserve (WR)strain and as A44L in Copenhagen strain encodes a protein that has 31%sequence identity to human 3β-hydroxy steroiddehydrogenase/Δ⁵-Δ⁴isomerase (hereinafter 3β-HSD) (Goebel et al. 1990,Blasco et al., 1991, and Smith et al., 1991). The SalF7L/A44L(hereinafter referred to as A44L) gene product was shown to haveactivity as a 3β-HSD by the conversion of pregnenolone to the steroidhormone progesterone (Moore and Smith, 1992). Deletion of the gene wasshown to abrogate virus 3β-HSD activity and reduce virus virulence. Forexample, mice infected with VV WR lacking A44L showed reduced mortalityand a milder weight loss compared to those infected with wild-type VVWR.

Various roles for the A44L 3β-HSD in VV virulence have been proposed.Proposals include that the steroid hormone product increases themetabolic rate, increases cell proliferation or causesimmunosuppression. None of those effects has yet been proven.

The role of A44L in VV virulence and immunogenicity was investigated bySroller (Sroller et al., 1998). It was observed that only moderateattenuation of virulence was achieved by deletion of A44L and thatimmunogenicity of the VV was not affected. It was concluded that a rolefor A44L in immunosuppression was unlikely as far as the humoral immuneresponse is concerned.

3β-HSD Enzymes

The 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴isomerase (3β-HSD) isoenzymesplay a key role in cellular steroid hormone synthesis. The 3β-HSD enzymecatalyses the conversion of Δ⁵-3β-hydroxysteroids to Δ⁴-3-ketosteroids,a reaction that is required for the biosynthesis of all classes ofsteroid hormones: progesterone, mineralocorticoids, glucocorticoids(GCs), androgens and estrogens. The enzyme is expressed in classicalsteroidogenic tissues (adrenal cortex, ovaries and testis) but also inperipheral tissues including the skin, liver, kidney and lung (Labrie etal., 1992; Martel et al., 1994; Pelletier et al., 1992). Multipleisoforms of 3β-HSD have been identified in human, rat and mouse tissues(Morel et al., 1997).

Steroids play an important role in the differentiation, growth andphysiology of many mammalian tissues. In addition, steroids havemultiple effects within the immune system. GCs are potentimmunosuppressive and anti-inflammatory agents and are usedtherapeutically in the treatment of organ transplantation, autoimmunedisease and a broad spectrum of inflammatory diseases (Boumpas et al.,1993). Although the precise mechanisms underlying theirimmunosuppressive effects have not been fully elucidated, GCs affect theimmune system by modulating cytokine production, antigen presentation,and the migration and cytotoxicity of immune cells (Ashwell et al.,2000; Boumpas et al., 1991; Schwiebert et al., 1996). In addition, sexhormones are reported to modulate immune responses and have beenimplicated in sex-associated susceptibilities to infectious agents, forexample coxsackievirus (Huber et al., 1999; Huber and Pfaeffle, 1994).GCs appear to have the potential to affect multiple aspects of theantiviral immune response.

GC release through the hypothalamic-pituitary-adrenal (HPA) axis occursas part of the circadian rhythm (Dhabhar et al., 1994) and is induced byadditional stimuli including physical or cognitive stress (Khansari etal., 1990) and cytokine responses to bacterial LPS (Fong et al., 1989;Imura and Fukata, 1994) or viruses such as murine cytomegalovirus (MCMV)(Ruzek et al., 1997).

Steroid hormones such as GCs have long been known to affect thepathogenesis of bacterial, protozoan and viral infections (Kass andFinland, 1953). A natural example of this is the severity of variolavirus infections observed in pregnant women, who were more likely toexhibit the fatal haemorrhagic form of smallpox than were men ornon-pregnant women (Rao et al., 1963), presumably due to the effect ofpregnancy hormones. This could be partially mimicked by theadministration of steroids such as cortisone to variola virus-infectedmacaque monkeys (Rao et al., 1968). Cortisone administration has alsobeen shown to increase the severity of the primary inoculation lesionand to delay healing in guinea pigs (Kligman, 1951) and rabbits (Bugbeeet al., 1960) infected with VV. Early leukocyte infiltration was alsoreduced in rabbits inoculated with VV (Bugbee et al., 1960).

Biochemically, mammalian 3β-HSD functions at multiple steps in steroidbiosynthesis. While GCs are generally considered to beimmunosuppressive, other steroids such as androstenediol (a metaboliteof dehydroepiandrosterone (DHEA)) have been shown to augment immuneresponses to infections with coxsackievirus (Loria and Padgett, 1992),influenza virus (Padgett et al., 1997) or herpes simplex type 1 virus(Daigle and Carr, 1998).

A number of proteins are secreted from poxvirus-infected cells that canbind and inhibit specific components of the host immune system includingIFNs, complement, cytokines and chemokines (Alcamí and Koszinowski,2000). A44L is an intracellular v3β-HSD encoded by VV strain WR thatincreases virulence in vivo following intranasal (Moore and Smith, 1992;Sroller et al., 1998) infection of mice.

SUMMARY OF THE INVENTION

The invention provides, inter alia, a recombinant poxvirus, wherein thepoxvirus genome does not comprise a functional gene encoding a3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴isomerase, for use as a medicament.The invention also provides a recombinant poxvirus having a genomecomprising a non-poxvirus gene or a fragment of a non-poxvirus genewhich gene or fragment encodes an antigen, wherein the poxvirus genomedoes not comprise a functional gene encoding a 3β-hydroxysteroiddehydrogenase/Δ⁵-Δ⁴isomerase, for use as a medicament.

Preferably, the recombinant poxvirus of the invention is for use as avaccine.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of an assay of 3β-HSD activity in cellsinfected with various VV strains;

FIG. 2 shows the attenuation of VV infection by the deletion of A44L;

FIG. 3 shows the titres of vA44L (a wild-type VV expressing gene A44L),vΔA44L (a VV mutant lacking 86% of the A44L gene) and A44L-rev (arevertant virus in which the A44L gene has been re-inserted at itsnatural site into the vΔA44L deletion mutant) VV in the lungs (A),brains (B), spleens (C) and livers (D) of mice infected with thoseviruses;

FIG. 4 shows a characterization of bronchoalveolar lavage (BAL) cellsuspensions from mice infected with vA44L, vΔA44L or vA44L-rev VV;

FIG. 5 shows an analysis of lymphocytes from BAL of VV-infected mice;

FIG. 6 shows the production of interferon (IFN)-γ in the lungs ofVV-infected mice;

FIG. 7 shows the activity of virus-specific cytotoxic T lymphocytes(CTLs) in the lung of VV-infected mice.

FIG. 8 shows corticosterone levels in plasma and lungs after intranasalinfection with vv.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the observation that the A44L gene of VVinterferes with events in the host cellular response to VV infection.The applicants have now found that the absence of A44L in VV isassociated with relatively mild weight loss and signs of illness, aswell as reduced virus titres in lungs and secondary sites of virusreplication (brain, spleen and liver). The absence of A44L is alsoassociated with a more vigorous inflammatory response characterised bymore rapid recruitment of CD4⁺ and CD8⁺ lymphocytes, enhanced IFN-γproduction and augmented CTL activity in the lungs of infected mice.Furthermore, levels of corticosterone, the natural murine glucocorticoid(GC), were lower in the plasma and lungs of mice infected with VVlacking A44L in comparison with VV with A44L suggesting that enhancedsteroid production by the A44L protein contributes to immunosuppressionduring VV infection.

The interference by A44L reduces the host's cellular immune response toVV, thus allowing protracted virus replication and improved virusdissemination in the host. Conversely, removal of the A44L gene leads toan increase in host cellular immune response to VV infection. In avaccine, a strong host cellular immune response is desirable ifeffective immunity is to be developed.

The invention provides a recombinant poxvirus, wherein the poxvirusgenome does not comprise a functional gene encoding a 3β-hydroxysteroiddehydrogenase/Δ⁵-Δ⁴isomerase, for use as a medicament. Preferably, therecombinant poxvirus of the invention is for use as a vaccine against adisease caused by a poxvirus.

Preferably the recombinant poxvirus is an orthopoxvirus or a derivativethereof. More preferably, the recombinant poxvirus is a VV, a cowpoxvirus, a camelpox virus or an ectromelia virus or a derivative of any ofthose viruses. Most preferably, the recombinant poxvirus is a VV. A VVmay be a VV strain selected from the group consisting of Lister,Copenhagen, Wyeth, New York City Board of Health, NYVAC, Praha virus,DRYVAX Wyeth-derived virus, LIVP, IHD-J, IHD-W, Tian Tan, Tashkent, KingInstitute, Patwadanger, EM-63, Evans, Bern, LC16m0 or MVA. Preferably,the recombinant poxvirus is a VV strain selected from the groupconsisting of MVA, Lister, Copenhagen or Wyeth.

The medicament may be for use as a vaccine against a disease caused byan orthopoxvirus infection in man, i.e., in a human. The disease causedby an ortho-poxvirus is especially one selected from the groupconsisting of smallpox, monkeypox and cowpox. The medicament isespecially for use as a vaccine against smallpox.

The recombinant poxvirus according to the invention also findsapplication in the veterinary field. The medicament may be for use as avaccine against a disease caused by an orthopoxvirus infection in ananimal, in particular in a mammal, for example, a non-rodent mammal. Theanimal may be, for example, a companion animal, an animal used in animalhusbandry, or an animal used in sport or for transport, for example, acat or dog, a member of the cattle family, a sheep or goat, a pig, ahorse, or a member of the camel family. The disease caused by anorthopoxvirus in an animal is especially one selected from the groupconsisting of monkeypox, cowpox, and camelpox. For example, themedicament may be for use in the immunisation of cats against cat pox,mice against ectromelia, members of the camel family against camelpox, awide range of mammals, for example rodents, cats, cows and other cattle,large felines or elephants against cowpox, or rodents or monkeys againstmonkeypox.

Alternatively, the recombinant poxvirus of the invention may be selectedfrom the group consisting of parapoxviruses, avipoxviruses,suipoxviruses, molluscipoxviruses and yatapoxviruses. Sequences ofcapripoxviruses and leporipoxviruses that have been determined to datedo not indicate the presence of a gene encoding a 3β-HSD. The amino acidsimilarity of the 3β-HSD enzymes between the genera is not particularlyhigh, typically 40-45% identity. For the definition of the term “geneencoding a 3β-HSD” used herein, see below.

Preferably, such a recombinant poxvirus of the invention may be for useas a vaccine against a disease caused by a poxvirus, for example amolluscum contagiosum virus, infection in man, that is to say, in ahuman.

Alternatively, such a recombinant poxvirus of the invention may be foruse as a vaccine against a disease caused by a poxvirus infection in ananimal, especially a mammal, wherein the poxvirus is selected from thegroup consisting of parapoxviruses, avipoxviruses, suipoxviruses,molluscipoxviruses and yatapoxviruses. The animal may be, for example,as described above, for example, a non-rodent mammal. The animal may be,for example, a companion animal, an animal used in animal husbandry, oran animal used in sport or for transport, for example, a cat or dog, amember of the cattle family, a sheep or goat, a pig, a horse, or amember of the camel family.

Optionally, the recombinant poxvirus of the invention has a genomecomprising a non-poxvirus gene or a fragment of a non-poxvirus genewhich gene or fragment encodes an antigen.

The invention further provides a vaccine composition comprising apoxvirus according to the invention and a pharmaceutically suitablecarrier.

The vaccine composition may comprise one or more additives selected fromthe group comprising an antibiotic, a preservative, a stabiliser and anadjuvant. Preferably, the vaccine composition according to the inventioncomprises one or more additives, for example a preservative and/or astabiliser. The immunising effect of an immunogen in a vaccine may beenhanced by the addition of an adjuvant. An adjuvant co-stimulates theimmune system in an unspecific manner causing a stronger specific immunereaction against the immunogenic determinant in the vaccine. Theinvention also provides a vaccine kit comprising a vaccine compositionaccording to the invention.

The invention further provides a method of vaccinating a subjectcomprising administering to the subject an effective amount of animmunogenic agent, wherein the immunogenic agent is a poxvirus accordingto the invention or a vaccine composition according to the invention.The vaccination generally induces an immune response to the poxvirusused as immunogenic agent and hence provides protection againstinfection by the poxvirus or an immunogenically cross-reacting poxvirus.

The term “subject” is used herein to denote a human or a non-humananimal. A non-human animal is, in particular, a mammal and may be anon-rodent mammal. The animal may be, for example, a companion animal,an animal used in animal husbandry or an animal used in sport or fortransport, for example, a cat or dog, a member of the cattle family, asheep or goat, a pig, a horse, or a member of the camel family.

The term “effective amount” denotes an amount effective to achieve thedesired result.

The invention also provides the use of a recombinant poxvirus having agenome which does not comprise a functional gene encoding a3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴isomerase for the manufacture of avaccine for the immunoprophylaxis of an infection caused by a poxvirus.

The terms “functional gene encoding a 3β-hydroxysteroiddehydrogenase/Δ⁵-Δ⁴isomerase” and “functional gene encoding a 3β-HSD”are used herein to mean a gene that gives rise to a gene product thathas effective 3β-HSD activity.

A 3β-HSD, or 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴isomerase is anyenzyme that catalyses the conversion of a Δ⁵-3β-hydroxysteroid to aΔ⁴-3-ketosteroid, irrespective of the amino acid sequence of the enzyme.The 3β-HSD activity in infected cells may be assessed, for example,using the tritiated pregnenolone method described by Moore and Smith(Moore and Smith, 1992). A level of activity in the assay statisticallysignificantly higher than that in an uninfected cell is considered to be“effective” and hence is the activity level above which a gene encodinga 3β-HSD is considered to be “functional” for present purposes.Preferably, an effective level of 3β-HSD activity is more than twice thelevel in an uninfected cell. More preferably, an effective level of3β-HSD activity is more than four times the level in an uninfected cell.

The absence of a “functional gene encoding a 3β-HSD” may be broughtabout by the poxvirus having no coding sequence for a 3β-HSD.Alternatively, it may be brought about by the poxvirus 3β-HSD genecoding sequence being disrupted, mutated or truncated such that its geneproduct has reduced activity or no activity. For example, 86% of the ORFmay be lacking. As a further alternative, one or more mutations ordeletions in the promoter or other upstream sequences may causeexpression of the gene to be compromised, leading to reduced levels ofgene expression or no gene expression.

Preferably, the absence of a functional gene encoding a 3β-HSD isbrought about by removal or disruption of a gene encoding a 3β-HSD in avirus, the parental strain of which comprises a functional gene encodinga 3β-HSD.

An example of a gene encoding a 3β-HSD is the A44L gene of VV strainCopenhagen. The sequence of the A44L gene is given in the completesequence of VV strain Copenhagen (Genbank Ref NC_(—)001559 and Goebel etal., 1990). The A44L gene of VV strain Copenhagen is in the Genbanksequence under accession number gi:9790357. The A44L ORF in VV strain WRis located in the F fragment of the SalI-restriction map of the virusgenome and hence was originally designated as SalF7L. Hereinafter it isreferred to as A44L (according to the HindIII-restriction map) in linewith VV strain Copenhagen (Goebel et al., 1990).

Genes that encode, or that have putatively been assigned as encoding, a3β-HSD have been identified in the genomes of other orthopoxviruses andin chordopoxviruses of genera other than orthopoxvirus. Only in thecases of the capripoxviruses and the leporipoxviruses sequenced to datehas a gene encoding a 3β-HSD not been found in a parental poxvirusgenome. The degree of amino acid identity between the proteins encodedby genes considered or shown to encode a 3β-HSD in the various generaand the A44L gene of VV strain WR is somewhat variable. Typically, thesequence of a 3β-HSD has amino acid identity of 25% or greater with thecoding sequence of the protein encoded by the A44L gene. For example,the sequence of a 3β-HSD has amino acid identity of 35% or greater withthe amino acid sequence of the protein encoded by the A44L gene. Mostpreferably, the sequence of a 3β-HSD has amino acid sequence identity of40% or greater with the sequence of the protein encoded by the A44Lgene.

By “derivative” of a particular virus is meant any virus that is derivedfrom the particular virus. A derivative may be obtained by repeatedpassaging of the particular virus. Alternatively, a derivative may beobtained by site directed or random mutagenesis of the particular virus.A derivative generally has most of the characteristics and most of thegenes of the particular virus from which it is derived. Typically, itsgenome has 90% sequence identity with the genome of the particularvirus. For example its genome has 95%, optionally 98% sequence identitywith the genome of the particular virus.

The invention also provides a recombinant poxvirus having a genomecomprising a non-poxvirus gene or a fragment of a non-poxvirus genewhich gene or fragment encodes an antigen, wherein the poxvirus genomedoes not comprise a functional gene encoding a 3β-hydroxysteroildehydrogenase/Δ⁵-Δ⁴isomerase for use as a medicament.

Preferably, the recombinant poxvirus of the invention is one wherein thepoxvirus is an orthopoxvirus or a derivative thereof. Preferably therecombinant poxvirus is one wherein the poxvirus is a VV, a cowpoxvirus, a camelpox virus or an ectromelia virus, or a derivative of anyof those viruses. Most preferably, the recombinant poxvirus is a VV. AVV strain may be selected from the group consisting of Lister,Copenhagen, Wyeth, New York City Board of Health, NYVAC, Praha virus,DRYVAX Wyeth-derived virus, LIVP, IHD-J, IHD-W, Tian Tan, Tashkent, KingInstitute, Patwadanger, EM-63, Evans, Bern, LC16m0 and MVA. For example,the VV strain is selected from the group consisting of MVA, Lister,Copenhagen or Wyeth.

Alternatively, the recombinant poxvirus of the invention may be selectedfrom the group consisting of parapoxviruses, avipoxviruses,suipoxviruses, molluscipoxviruses and yatapoxviruses.

The non-poxvirus gene or gene fragment that encodes an antigen may beany non-poxvirus gene or gene fragment against the gene product of whicha protective immune response in a subject is desirable.

By “non-poxvirus gene” is meant herein a gene not belonging to apoxvirus of the genus of the poxvirus in question. For a givenrecombinant poxvirus, the non-poxvirus gene may be a gene belonging to apoxvirus of a different genus. Preferably, the non-poxvirus gene is agene not belonging to any poxvirus.

The recombinant poxvirus of the invention is preferably for use as avaccine for the prophylaxis of an infection caused in a subject by apathogenic agent.

Preferably, the gene or gene fragment that encodes an antigen may be anynon-poxvirus gene or gene fragment against the gene product of which acellular immune response in a subject is desirable. Suitable genesinclude those encoding immunogenic peptides or polypeptides of aninfectious pathogen, for example, for use in humans, an influenza virus,malaria, HIV, heptitis C virus, hepatitis B virus, herpes virus, aparasitic pathogen, for example tuberculosis or Leishmaniasis, aprotozoan, for example a protozoan that causes ameobic dysentery. Foruse in animals, appropriate pathogen immunogenic peptides are known tothose skilled in the art.

The recombinant poxvirus of the invention may be for use as a vaccinefor the prophylaxis or treatment of a disease associated with aberrantcells. In such a recombinant poxvirus, the gene or gene fragment thatencodes an antigen may be any non-poxvirus gene or gene fragmentencoding an antigenic peptide or an antigenic polypeptide of aberrantcells, for example cancer cells, the elimination or induced quiescenceof which is beneficial.

In some instances, it is beneficial for the whole gene that encodes anantigen to be present in the virus. In some cases, however, a fragmentof the gene will suffice. In the case of a gene fragment, a gene productsmaller than that of the whole gene is produced, which smaller geneproduct is or comprises an epitope or epitopes of the antigen inquestion.

The invention also provides a vaccine composition comprising a poxvirusof the invention and a pharmaceutically suitable carrier. The vaccinecomposition may optionally comprise an adjuvant.

The invention further provides a method of vaccinating a subjectcomprising administering to the subject an effective amount of animmunogenic agent, wherein the immunogenic agent is a poxvirus accordingto the invention or a vaccine composition according to the invention.The vaccination generally induces an immune response to the poxvirusused as immunogenic agent and hence provides protection againstinfection by the poxvirus or an immunogenically cross-reacting poxvirus.

The invention further provides a method of inducing a protective immuneresponse in a subject comprising administering to the subject animmunogenic agent, wherein the immunogenic agent is a poxvirus or avaccine composition according to the invention. Preferably, the immuneresponse includes a cellular immune response. A protective immuneresponse is induced to the poxvirus used as immunogenic agent and henceprovides protection against infection by the poxvirus or animmunogenically cross-reacting poxvirus.

The invention also provides the use of a recombinant poxvirus accordingto the invention for the manufacture of a vaccine for the prophylaxis ofan infection caused by a pathogenic agent wherein said poxvirus has agenome comprising a non-poxvirus gene or a fragment of a non-poxvirusgene which gene or fragment encodes an antigen of the pathogenic agent.

The invention also provides the use of a recombinant poxvirus accordingto the invention for the manufacture of a vaccine for the prophylaxis ortreatment of a disease associated with aberrant cells, wherein saidpoxvirus has a genome comprising a non-poxvirus gene or a fragment of anon-poxvirus gene which gene or fragment encodes an antigen of theaberrant cells comprising the gene product of the said non-poxyiralgene.

A recombinant poxvirus of the invention may be prepared by methods knownin the art (see for example Boyle, D. B. and Coupar, B. E. H., Gene,1988, 65, 123-128). For example, a poxvirus lacking a functional genethat encodes a 3β-HSD may be produced by transfection of cells that hadpreviously been infected with a poxvirus with a plasmid, the plasmidcomprising DNA sequences homologous to sequences of the poxvirusflanking the gene that encodes a 3β-HSD or in the gene that encodes a3β-HSD together with a selectable marker. After transfection and virusmultiplication, recombinant viruses are selected using the selectablemarker.

As mentioned above, the poxvirus of the invention may comprise a genethat encodes an immunogen. The gene encoding the antigen is introducedinto the poxvirus in a manner known in the art. For example, a plasmidmay be used that comprises the gene together with DNA sequenceshomologous to sequences of the poxvirus genome such that homologousrecombination can take place between the plasmid and the genomic DNA.Preferably, the sequence of the poxvirus genome is one that may bedisrupted without compromising the viability of the poxvirus. Thehomologous DNA sequences are preferably of sufficient length to enablehomologous recombination between the plasmid DNA and the virus genomicDNA to take place. Accordingly, the DNA sequences preferably have alength of from 20 to 1000 bp. More preferably, the sequences have alength of from 100 to 800 bp. Still more preferably, the sequences havea length of from 300 to 500 bp. Sequences are considered homologous ifthey have 85% or more sequence identity. Preferably, homologoussequences have 90% or more sequence identity, more preferably,homologous sequences have 95% or more sequence identity, for example,98% or more sequence identity. Most preferably, the homologous sequencesused are identical to the genomic sequences in the virus.

By use of a plasmid comprising the gene encoding the antigen flanked byDNA sequences with homology to sequences of the poxvirus flanking thegene that encodes a 3β-HSD or in the gene that encodes a 3β-HSD A44Lgene, it is possible to carry out the disruption of the gene thatencodes a 30-HSD and the introduction of the antigen gene in a singlestep.

The gene encoding the antigen of interest is preferably engineered to beassociated with transcriptional regulatory sequences for expression ofthe gene by poxvirus in an infected host cell. Such regulatory sequencespreferably include a promoter from a poxvirus and a poxvirus terminationsequence. Most preferably, a promoter from VV or a related poxvirus anda VV termination sequence are included. Most preferably, the VVtermination sequence is a VV early transcriptional termination sequence(TTTTTNT).

The invention further provides a recombinant poxvirus having a genomecomprising a non-poxvirus gene or a fragment of a non-poxvirus genewhich gene or fragment encodes an antigen, wherein the poxvirus genomedoes not comprise a functional gene encoding a 3β-hydroxysteroiddehydrogenase/Δ⁵-Δ⁴isomerase, with the proviso that the non-poxvirusgene or fragment of a non-poxvirus gene is not a gene encodingvaricella-zoster virus glycoprotein E, hepatitis B virus preS2-S proteinor E. coli guanine phosphoribosyl transferase.

For the preparation of a vaccine, the poxvirus according to theinvention is typically provided in a physiologically acceptable form. Aperson skilled in the art will be familiar with suitable poxvirusvaccine formulations given the large body of knowledge that was built upin the many years of use of VV in the vaccination against smallpox. Forexample, an appropriate number of particles of the recombinant poxvirus,e.g. 10⁴ to 10⁹ particles, are freeze dried in an appropriate volume,e.g. approximately 100 μl, of phosphate-buffered saline (PBS) in thepresence of peptone and human albumin in, for example, a vial or anampoule, preferably a glass ampoule. The lyophilisate can containextenders, for example mannitol, dextran, sugar, glycine, lactose orpolyviylpyrrolidone, or other excipients, for example antioxidants orstabilisers, suitable for parenteral administration. The vial or ampoulemay then be sealed and may be stored for several months, preferably at atemperature below −20° C.

For vaccination, the lyophilisate may, for example, be made up to 0.1 to0.2 ml of aqueous solution, preferably with physiological saline, andadministered parenterally, for example by intradermal inoculation or bydermal abrasion. The vaccine of the invention may be infectedintracutaneously. The mode of administration, the dose and the number ofadministrations can be optimised by those skilled in the art in aconventional manner. A high degree of immunity against the antigen isobtained by administration of the vaccine several times over a lengthyperiod of time.

Biological Significance of A44L Protein in Cellular Immunity

The present invention provides a poxvirus lacking a functional gene thatencodes a 3β-HSD. An example of such a gene is the VV A44L gene. It hasbeen found by the present applicants that A44L is an important virulencefactor of VV and has potent immunosuppressive properties. The absence ofa functional A44L gene therefore results in the immune response to thevirus being enhanced. Investigations into the effects of a functionalA44L gene and the effects of the absence thereof are described below.

3β-HSD Activity of VV Lacking A44L and Virulence Thereof

3β-HSD Activity

Expression of 3β-HSD activity in infected cells by strains of VV wasmeasured in vitro by the conversion of [³H]-pregnenolone to[³H]-progesterone. As described in further detail in the Examplessection below, all normal parental VV strains investigated exhibited3β-HSD activity indicating v3β-HSD is highly conserved in VVs. Theactivity was not observed in strains of VV lacking a functional A44Lgene. Upon infection of host cells, a VV without the A44b gene was foundto generate a similar amount of infectious progeny virus as a VV withA44L. It appears unlikely, therefore, that the different levels of3β-HSD activity seen reflect differences in the amount of virus present.

Effect of deletion of A44L on VV In Vivo

The role of the A44L protein in VV infection may be explored usingvarious models of infection. The present inventors used a murineintranasal model. In the model, mice are infected with VV and thenweighed individually and assessed for general signs of illness dailyover a period of approximately 2 weeks.

Using such a method, the present inventors found that the infectionphenotype is attenuated in mice infected with VV lacking A44L relativeto parental and revertant viruses. Mice infected with parental orrevertant VV generally lost over 20% of body weight during the infectionand displayed severe signs of illness manifested by ruffled fur, reducedmobility and tachypnea. In contrast, mice infected with VV lacking A44Llost significantly less weight, recovered more rapidly and displayedvery mild clinical signs of illness.

A further measure of infection progression is the change in bodytemperature with time after infection. To determine if A44L could affectbody temperature during VV infection, rectal temperatures in mice weremeasured on a daily basis following infection with VV. No differenceswere seen in the early body temperatures (days 1 to 5 post infection(p.i.)) recorded in animals infected with VV lacking A44L and in animalsinfected with parental VV.

The B15R ORF of VV strain WR encodes a soluble IL-1β receptor and it wasreported by Alcamí and Smith (Alcamí and Smith, 1992) that deletion ofB15R results in a more virulent infection following intranasalinoculation of mice. Animals infected with VV lacking B15R displayedelevated temperatures at early times of infection suggesting that theB15R protein blocks IL-1β-induced fever (Alcamí and Smith, 1996). Thefact that no such temperature effect was seen in the case of infectionwith VV lacking A44L indicates that A44L affects virulence through analternative mechanism(s).

Previously, it had been reported that mice infected intranasally withvΔA44L (a recombinant VV WR strain lacking A44L but not comprising anexpressed non-poxvirus gene or an expressed fragment of a non-poxvirusgene which gene or fragment encodes an antigen showed reduced mortalityand relatively mild weight loss compared to those infected with parentalstrain WR (Moore and Smith, 1992). In a follow up study, Sroller et al.(Sroller et al., 1998) reported that loss of A44L had only a relativelymodest effect on virulence following intranasal infection of mice withVV strain WR. They reported observing that only moderate attenuation ofvirulence was achieved by deletion of A44L and that immunogenicity ofthe VV was not affected. Deletion of the v3β-HSD from VV did not enhanceantibody responses to infection and it was concluded that a role forA44L in immunosuppression was unlikely.

Despite the results reported by Sroller et al., the present inventorscontinued to investigate the immune response to VV strain WR lacking afunctional A44L gene. The present inventors have found that thecell-mediated immune response is influenced by the presence or absenceof A44L. The cell-mediated immune response is essential if effectiveimmunity is to be achieved against certain types of pathogen.

A44L is non-essential for virus replication in vitro. However it appearsto be well conserved amongst strains of VV and some other members of thepoxvirus family. All VV strains tested expressed 3β-HSD activity andsimilar activity has been described in cells infected with VV strainsPraha, LIVP and MVA (Sroller et al., 1998) as well as the avipoxvirusesfowlpox and canarypox (Skinner et al., 1994) and a fish iridiovirus(Baker and Blasco, 1992). The gene is also highly conserved in severalother sequenced orthopoxviruses (camelpox, ectromelia, cowpox andmonkeypox) and other poxvirus genera (suipoxvirus, molluscipoxvirus andyatapoxvirus) suggesting the v3β-HSD plays an important role in poxvirusbiology, perhaps in antiviral host defence. Interestingly, despite thepresence of an A44L-like gene in several variola strains, in each casethe gene has been disrupted by mutation and is not predicted to encodean active A44L counterpart (Aguado et al., 1992; Massung et al., 1993;Massung et al., 1994; Shchelkunov et al., 1995; Shchelkunov et al.,2000).

Clearance of VV Lacking A44L

Spread of VV in the Lung

In the intransal model for VV infection, the lung is the site of primaryinfection. As described in further detail in the Example section below,VV with or without A44L was found to establish infections in the lungand produce similar virus titres at 1 and 3 days p.i., indicating thatA44L is not critical for the initial rounds of replication in the lung.However, by the time peak titres of virus occur, typically at 7 daysp.i., the level of infectivity in the lungs was significantly greater inmice infected with parental VV than those infected with VV lacking A44L.By day 12, infectious virus was only detectable in a minority of animalsinfected with VV lacking A44L compared with all, or a substantialmajority, animals infected with parental VV.

Spread in Cells Other than Lung Cells

To examine the ability of VVs to spread and replicate in extrapulmonarysites, homogenates of brain, spleen and liver of infected mice wereassayed for infectious virus. In infected mice, virus titres were foundto be below detection levels in all organs at day 1 p.i., however by day3 p.i. infectious virus was recovered from the spleen and livers of allmice infected with W with or without A44L. By day 3 p.i. infectiousvirus was recovered from the brains of most of the mice infected with VVwith A44L but from only a minority of mice infected with VV lackingA44L.

By day 7, virus titres were reduced in all of the organs examined frommice infected with VV lacking A44L, indicating that while this virus iscapable of in vivo spread, it is cleared more rapidly from the brain,liver, spleen and lungs compared to parental VV.

To investigate the replication of VV in murine cells in vitro, culturesof primary fibroblasts may be prepared from the kidneys of naive BALB/cmice. The replication kinetics were analysed according to single-step(10 PFU/cell) or multistep (0.01 PFU/cell) growth curves, the latterconditions being particularly sensitive to accentuating smalldifferences in growth between viruses. VV with and without A44L werefound to have similar replication kinetics.

In conclusion, it has also been found that although

-   (i) VV with or without A44L replicates to similar levels in primary    murine fibroblasts,-   (ii) virus titres in the lungs of mice infected with VV with or    without A44L were equivalent on day 1 p.i., and-   (iii) VV with or without A44L were capable of spread and replication    in secondary organs (brain, liver, spleen), the virus lacking A44L    was cleared more rapidly. These findings indicate that A44L is not    critical for the initial rounds of virus replication in the host and    that the difference between the viruses is likely to be due to host    antiviral mechanisms rather than altered replicative ability.    Inflammatory Response to VV Lacking A44L

The observation that mice infected with VV lacking A44L were able toclear virus rapidly suggested a more effective antiviral host responseagainst that virus. Therefore, the cellular inflammatory response toinfection with VV in the lung was assessed.

BAL Cells from Lungs of VV-Infected Mice

BALs were performed as described in the Examples section below. Only fewcells were recovered from BAL of animals 1 day post infection (p.i.)with VV, indicating there were only low levels of recruitment to thelung in the early phase of VV infection. BAL cell numbers increased overtime with peak numbers 12 days p.i. for VV with A44L, and 7 days p.i.for VV lacking A44L. Significantly more cells were recovered at day 7p.i. for mice infected with VV lacking A44L compared with mice infectedwith VV with A44L.

BAL cells were centrifuged onto slides for microscopic examination anddifferential cell counts were carried out. Uninfected mouse BAL cellswere composed almost entirely of macrophages, with some (<10%)lymphocytes noted. The majority of inflammatory cells recruited to thelung during the course of VV infection were macrophages and lymphocytes,with granulocytes representing less than 5% of total cells at all timepoints tested.

Recruitment of lymphocytes to the lungs at day 7 was found to bestatistically enhanced in mice infected with VV lacking A44L. Thatobservation correlates with the more rapid clearance of that virus fromthe lungs of infected animals. Notably, for mice infected with VVlacking A44L by day 12 after infection the levels of total cells,macrophages and lymphocytes in BALs were all lower than on day 7,whereas for mice infected with VV containing A44L the cell numbers werehigher on day 12 than on day 7.

A striking feature of the cellular inflammatory response to infectionwith VV lacking A44L was the early recruitment of lymphocytes to thelung. Flow cytometric analysis was used to examine lymphocyte subsets inBAL at day 7 p.i. A greater percentage of both CD4⁺ and CD8⁺ lymphocyteswere observed in BAL from mice infected with VV lacking A44L compared toanimals infected with VV with A44L. Less than 5% of BAL cells were DX5⁺natural killer (NK) cells or B220⁺ B lymphocytes at this time. By day 7p.i. with VV lacking A44L more cells were recruited to the lungs and ahigher proportion of those were CD4⁺ and CD8⁺ T cells.

IFN-γ Production in Lungs of VV-Infected Mice

The results discussed indicate an increased presence of CD4⁺ and CD8⁺ Tcells within the lungs of mice infected with VV lacking A44L.

IFN-γ plays an important role in antiviral host defence followinginfection of mice with VV or other poxviruses (Dalton et al., 1993;Huang et al., 1993; Muller et al., 1994). Given the marked enhancementin T cell recruitment to the lungs of mice infected with VV lacking A44Land the ability of both CD4⁺ and CD8⁺ T lymphocytes to produce antiviralcytokines, the levels of IFN-γ in BAL fluids from VV-infected mice weredetermined.

Low levels of IFN-γ were detected in lavage fluids 3 days p.i. and thoseincreased markedly at days 7 and 10 p.i. Significantly higher levels ofIFN-γ were detected in lavage fluids from mice infected with VV lackingA44L compared to animals infected with control parental viruses at day 7p.i. IFN-γ levels at day 10 were also generally higher in mice infectedwith VV lacking A44L. Levels of IL-10 or IL-4 in BAL fluid were belowthe detection limit of their respective ELISA assays at each time pointstested.

IFN-γ production was also determined after culturing lung cellsuspensions in the presence of PMA and ionomycin. It was found thatIFN-γ levels were very low in lung cell supernatants from mock-infectedmice. A modest increase was observed in supernatants from lung cells 3days p.i., and levels were elevated further from lung cells harvestedday 7 and day 10 p.i. Consistent with IFN-γ levels observed in BAL,PMA-stimulated lung cells from mice infected with VV lacking A44Lproduced significantly more IFN-γ on day 7 and 10 than those fromanimals infected with VV with A44L p.i.

The elevated IFN-γ production detected in BAL fluids and lung cellsuspensions from vΔA44L-infected mice suggests that the additionallymphocytes recruited to the lungs of these animals were producingIFN-γ. Intracellular staining of lung lymphocytes for IFN-γ on days 3, 7and 10 p.i. showed that the proportion of both CD4⁺ and CD8+ lymphocytespositive for IFN-γ was higher at days 7 and 10 p.i. in the lungs of miceinfected with VV lacking A44L.

Only a very low proportion of CD4⁺ or CD8⁺ cells (<5%) stained for IL-4or IL-10.

Cytolytic Activity of Lymphocytes Isolated from VV-Infected Lungs

To assess the effect of VV A44L on virus-specific CTL activity, thecytotoxic activity of effector cells from lung cell suspensions may beexamined. At day 5, primary CTL activity was very low in lung cellsuspensions from all infected animals, however by day 7 significant CTLactivity was detected against virus-infected targets.

The cytolytic activity of lung lymphocytes of mice infected with VVlacking A44L was greater than from mice infected with VV with A44L, andthis trend was also observed using lung cells from animals 10 days p.i.

The increase in cytotoxic activity of lung cells from vΔA44L-infectedmice may represent increased recruitment of CTL effectors to the lung oran increased activation state of the lung cells present.

To assess whether the CD8⁺ T lymphocytes were mediating CTL activity,day 7 lung cells from VV-infected mice were treated with complement anda mAb to murine CD8. The treatment abrogated virtually all CTL activity.

The numbers of CD8⁺ lymphocytes in the lung cell suspensions weredetermined by flow cytometry, and used to compare CTL activity betweenlung cells based on a CD8+ lung cell:target ratio. The enhanced lysisobserved using lung cells from mice infected with VV lacking A44L ispartly explained by the higher relative numbers of CD8⁺ T cells in thiscompartment. However, CTL from mice infected with VV lacking A44L alsoshow an enhanced level of lysis on a per cell basis consistent with anenhanced activation state of virus-specific CTL within the T cellpopulation.

Levels of Corticosterone in Plasma and Lungs of VV-Infected Mice

It has been found by the present inventors that VV lacking A44L ismarkedly attenuated in the murine intranasal model, and that this isaccompanied by a more vigorous cellular inflammatory response in thelungs of infected mice. The A44L ORF encodes an active v3β-HSD enzymecapable of converting pregnenolone to progesterone (Moore and Smith,1992) as discussed in further detail in the introduction. As there isgrowing evidence that GCs and other steroids may possess importantimmunomodulatory functions, the levels of corticosterone were determinedin the plasma and lungs of VV-infected mice. Infection with VV inducedan increase in corticosterone levels in plasma and lung extracts asearly as 1 day p.i., and the levels increased further up to day 4.Levels were significantly lower in samples from mice infected with VVlacking A44L relative to mice infected with VV with A44L.

Corticosterone is released as part of the acute phase response toinfection and other inflammatory trauma. Viral titres in the lungs werenot significantly different between mice infected with VV with orwithout A44L at days 1, 2 and 4 p.i., nor were there differences invisible signs of illness or weight loss at these early time points. Theenhanced levels of endogenous GCs observed in mice infected with VVcontaining A44L suggest that A44L may have a direct effect upon localand systemic steroid levels during VV infection.

Conclusions

The rapid clearance of vΔA44L coincided with an early influx ofinflammatory leukocytes, in particular T lymphocytes, into the lung ofmice infected with VV lacking A44L.

Functional analysis suggests that IFN-γ production and virus-specificCTL activity are enhanced in lymphocytes present in the lungs of miceinfected with VV lacking A44L. That phenotype is consistent with thehypothesis that the A44L protein interferes with aspects of thepro-inflammatory host response.

Previously, the A44L gene from VV strain WR was shown to encode anactive 3β-HSD enzyme (Moore and Smith, 1992). In view of the prior artinformation available regarding 3β-HSD enzymes (see above) a possiblerole for A44L in VV pathogenesis is in the production of steroidhormones such as GCs.

Previous studies have shown that restraint stress (RST) reduced theaccumulation of leukocytes in the lungs of influenza virus-infected miceand that this response was dependent upon elevated serum GCs astreatment with the GC receptor antagonist RU486 restored cellularinfiltration (Hermann et al., 1995). RST suppression of influenzavirus-specific production of cytokines such as IL-2 and IFN-γ was alsoreversed by RU486 treatment (Dobbs et al., 1996). By analogy, thepresent findings that infection of mice with VV lacking A44L wasassociated with enhanced leukocyte infiltration and IFN-γ production inthe lung is consistent with A44L-mediated enhancement of GC levels invivo following infection with parental VV.

The increased levels of corticosterone in plasma and in the lungcompared to uninfected mice and the significant increase in plasma andlung corticosterone levels in mice infected with VV with A44L comparedto mice infected with VV lacking A44L are consistent with a directeffect of the A44L protein on increasing steroid levels in vivo.Alternatively, the differences observed might reflect the more severeillness caused by the VV with A44L as corticosterone is part of theacute phase response observed after infection or other inflammatorytrauma. It is worth noting, however, that there were no differences insigns of illness, nor in virus titres recovered from the lungs of miceinfected with VV with or without A44L at days 1, 2 and 4 p.i. whilecorticosterone levels differed at those times.

The mechanisms governing the immunomodulatory properties of the A44Lprotein in VV pathogenesis in vivo have not yet been established.Biochemically, mammalian 3β-HSD functions at multiple steps in steroidbiosynthesis and the A44L protein has been shown previously to becapable of converting at least two substrates, pregnenolone anddehydroepiandrosterone (DHEA) to their respective 5-ketosteriods. Thenature of the substrates available and the types and levels of differentsteroids produced in the lung following VV infection may provideimportant information as to the principal reaction/s catalysed in vivoby A44L.

A striking feature of the inflammatory response to VV lacking A44L isthe rapid recruitment of leukocytes to the lungs of virus-infected mice,in particular CD4⁺ and CD8⁺ T lymphocytes with the potential to produceIFN-γ and CD8+ CTLs, indicating that A44L can suppress at least twoimportant components of the antiviral host response.

Major histocompatability complex (MHC) class I-restricted, CD8⁺ CTL andthe antiviral cytokine IFN-γ are believed to play an important role inthe resolution of acute infection by poxviruses such as VV (Huang etal., 1993; Ramsay et al., 1993; Ruby and Ramshaw, 1991). VV is known tobe very sensitive to IFN-γ in vitro (Melkova and Esteban, 1994) andIFN-γ production is critical for recovery of mice after VV infection(Dalton et al., 1993; Huang et al., 1993; Karupiah et al., 1990; Rubyand Ramshaw, 1991). Poxviruses encode a number of proteins that areexpressed intracellularly and extracellularly to interfere with theantiviral effects of IFN-γ. However, a viral IFN-γ receptor secretedfrom VV-infected cells does not neutralize mouse IFN-γ (Alcamí andSmith, 1995; Mossman et al., 1995). CD8⁺ CTLs are key mediators of viralclearance by cytolysis of virus-infected cells and the secretion ofcytokines such as IFN-γ and TNF-α. Interestingly, GCs have also beenreported to suppress CTL activity (Schleimer et al., 1984).

It is apparent from the inventors' data presented above that A44L isimmunosuppressive and interferes with the early events in the hostcellular response to VV infection, thus contributing to protracted virusreplication and improved virus dissemination within the host. It followsthat the removal of the A44L gene from poxyvirus vaccines, for examplemodified virus Ankara (MVA), enhances vaccine immunogenicity,particularly T cell responses to infection.

EXAMPLES

Cells and Viruses

BS-C-1 (African green monkey, epithelial), CV-1 (African green monkey,fibroblast) and TK-143 (human osteosarcoma) cells were gown inDulbecco's modified Eagle's medium (DMEM) supplemented with 10%heat-inactivated foetal bovine serum (FBS). The origins of the VVstrains used herein were as described by Alcamí et al. (Alcamí et al.,1998). VV strain WR was grown and partially purified through sucrosecushions as described by Mackett et al. (Mackett et al., 1985). VVstrain WR is available from ATCC under number VR-119.

Construction of Recombinant Viruses

Wild-type VV strain WR is referred to hereinafter as vA44L in view ofits having a full viral A44L gene. Recombinant virus vΔA44L, in which86% of the A44L ORF of VV strain WR was replaced with the selectablemarker E. coli guanine phosphoribosyl transferase (Ecogpt), wasconstructed as described by Moore and Smith (Moore and Smith, 1992).

A revertant virus, based on vΔA44L and into which a functional A44L genehad been inserted was used as a control to ensure that any phenotypicdifferences of vΔA44L were due to loss of the A44L protein and not dueto mutations elsewhere in the genome.

Revertant virus vA44L-rev was constructed by replacing the Ecogpt ofvΔA44L with the A44L gene. Plasmid pJM5 was constructed by cloning a2318-bp HincII fragment of VV strain WR containing the A44L ORF andflanking regions into the unique SmaI site of pUC119. Plasmid pJM5 wastransfected into vΔA44L-infected cells and mycophenolic acid(MPA)-resistant recombinant viruses were isolated as described byFalkner and Moss (Falkner and Moss, 1990). These were grown onhypoxanthine guanine phosphoribosyl-transferase negative D980R cells inthe presence of 6-thioguanine and plaque isolates corresponding todeletion mutant or revertant viruses (A44L-rev) were identified.

Analysis of vA44L, vΔA44L and vA44L-rev viruses by PCR confirmed thatthe entire A44L gene was present in only vA44L and vA44L-rev viruses andthat the genes flanking the A44L locus were similar in each of the threeviruses. Southern blots of the DNA of the three viruses showed that thegenomes were as predicted. The plaque morphology formed on BS-C-1 cellsby vA44L, vΔA44L and vA44L-rev viruses was indistinguishable, confirmingthat loss of A44L does not affect virus replication in vitro.

3β-HSD Assay

Monolayers of CV-1 cells in 24-well plates were mock-infected orinfected in triplicate with VV at 10 PFU/cell. At 10 h p.i. 3β-HSDactivity was measured by conversion of [³H]-pregnenolone to[³H]-progesterone as described by Moore and Smith (Moore and Smith,1992). All infections were performed in triplicate and results areexpressed as mean±SEM. The background values from non-enzymaticconversion to progesterone (measured in ethanol-fixed monolayers) weresubtracted from each value. The data for vA44L, vΔA44L or A44L-rev areshown in FIG. 1A. As seen in that Figure, vA44L- and vA44L-rev-infectedCV-1 cells produced 3β-HSD activity but mock- and vΔA44L-infected cellsdid not.

To extend these findings, a range of VV strains were examined for theirability to produce 3β-HSD activity in vitro. Eight strains of VV wereassayed for 3β-HSD activity at 8-10 h p.i. The data for the eightdifferent VV strains are shown in FIG. 1B. All eight strains tested wereable to convert [³H]-pregnenolone to [³H]-progesterone, indicatingv3β-HSD is highly conserved in VVs.

Cells from duplicate wells were harvested during the experiment and theamount of infectious virus was found to be similar in all samples,indicating it is unlikely that the different levels of 3β-HSD activityseen reflect differences in the amount of virus present.

Assay for Virus Virulence

The role of the A44L protein in VV infection was explored using a murineintranasal model of infection. Groups of five 6 to 8 week-old femaleBALB/c mice infected with 10⁴ PFU of vA44L, vΔA44L or vA44L-rev. Micewere subjected to brief anesthesia and were inoculated intranasally with10⁴ or 10⁵ PFU of VV in PBS on day 0. Each day, mice were weighedindividually and the results are expressed as the mean percentage weightchange of each group±SEM compared with the weight immediately prior toinfection. Mice were also monitored for signs of illness, and they werescored from 1 to 4. Data from each day are expressed as the mean±SEMfrom 5 mice. Mice suffering a severe infection or having lost >25% oftheir original body weight were sacrificed. The change in weight and thesigns of illness seen in the mice in the days immediately afterinfection are shown in FIGS. 2A and 2B in which ⋄=mock-infected,▪=infected with vA44L, ◯=infected with vΔA44L and A=infected withA44L-rev.

It is seen in FIG. 2 that the infection phenotype is attenuated invΔA44L relative to both vA44L and vA44L-rev viruses. Mice infected withvA44L or vA44L-rev lost over 20% of body weight during the infection(FIG. 2A) and displayed severe signs of illness manifested by ruffledfur, reduced mobility and tachypnea (FIG. 2B). In contrast, miceinfected with vΔA44L lost significantly less weight, recovered morerapidly and displayed very mild clinical signs of illness. There was nosignificant difference between the weight loss profiles of mice infectedwith vA44L or vA44L-rev indicating that the attenuation seen with vΔA44Lis due to loss of the A44L gene.

P values were determined using the Student's t-test and indicate themean % weight changes or signs of illness of mice infected with vΔA44Lthat were significantly different from both those of mice infected withvA44L or A44L-rev.

Spread of VV in the Lung, Brain, Spleen and Liver

WR is a neurovirulent strain of VV that was derived by repeated passagein suckling mouse brain (Bronson and Parker, 1941). In the murineintranasal model, VV infection is accompanied by extensive respiratoryinfection and virus dissemination to multiple organs (Turner, 1967;Williamson et al., 1990). In the intranasal model for VV infection, thelung is the site of primary infection. To examine the ability of thethree viruses to spread and replicate in extrapulmonary sites,homogenates of brain, spleen and liver were also assayed for infectiousvirus.

Groups of 5 mice were infected intranasally with 10⁴ PFU of vA44L,vΔA44L or vA44L-rev and the lungs, brains, spleens and livers wereharvested at noted times p.i. After sacrifice of the mice, their lungs,brains, livers and spleens were removed, dounce homogenized, frozen andthawed three times and sonicated and stored at −70° C. The titre ofinfectious virus was determined by plaque assay on BS-C-1 cells. Thetitres of vA44L, vΔA44L and A44L-rev in the lungs (A), brains (B),spleens (C) and livers (D) are shown in FIG. 3. Virus titres areexpressed as mean log₁₀PFU per organ, with SEM. The broken lineindicates the minimum detection limit of the plaque assay. Columnsmarked with an asterisk represent virus titres from vΔA44L-infected micethat were significantly different to those from both vA44L- andvA44L-infected animals. *, P<0.05, **, P<0.02.

As seen in FIG. 3A, the three viruses established infections in the lungand produced similar virus titres at 1 and 3 days p.i., indicating thatA44L is not critical for the initial rounds of replication in the lung.However, by the time peak titres of virus occurred at 7 days p.i. thelevel of infectivity in the lungs of vA44L and vA44L-rev-infected micewas significantly greater than that in mice infected with vΔA44L. By day12, infectious virus could be detected in the lungs of only 1/5vΔA44L-infected animals compared to 5/5 in each of the other groups.

The results of the brain, spleen and liver experiments are shown inFIGS. 3B, C and D respectively. Virus titres were below detection levelsin all organs at day 1 p.i., however by day 3 p.i. infectious virus wasrecovered from brain, spleen and liver of all mice infected with vA44Lor vA44L-rev, and from the spleen and livers of all vΔA44L-infectedanimals. Infectivity titres were similar in all groups at this time,except in the brain of vΔA44L-infected mice, where virus was recoveredfrom only 1/5 animals. By day 7, virus titres were reduced in all of theorgans examined from vΔA44L-infected mice, indicating that while thisvirus is capable of in vivo spread, it is cleared more rapidly from thebrain, liver, spleen and lungs compared to vA44L and vA44L-rev viruses.Virus was still present in lungs of VV-infected animals 12 days p.i.,however it had been cleared from all other organs examined by this time.

To investigate if vA44L, vΔA44L and vA44L-rev could replicate to similarlevels in murine cells in vitro, cultures of primary fibroblasts wereprepared from the kidneys of naive BALB/c mice. The replication kineticsof the three viruses were similar during single-step (10 PFU/cell) ormultistep (0.01 PFU/cell) growth curves; the latter conditions areparticularly sensitive to accentuating small differences in growthbetween viruses.

Recovery of Bronchoalveolar Lavage (BAL) and Lung Cells

Mice were infected with 10⁴ PFU of vA44L, vΔA44L or vA44L-rev. BAL fluidwas obtained from mock- and VV-infected mice at various times p.i. Micewere sacrificed and the lungs of each mouse were inflated five timeswith a 1 ml volume of PBS containing 10 U/ml of heparin through ablunted 23-guage needle inserted into the trachea. BAL was centrifugedat 3,000 rpm for 10 min and the supernatant was removed and frozen at−20° C. for analysis of cytokines by ELISA. BAL cells were treated withTris-NH₄Cl (0.14 M NH₄Cl in 17 mM Tris, adjusted to pH 7.2) to lyseerythrocytes, washed twice and resuspended in cold RPMI 1640 mediumsupplemented with 10% FBS. BAL cells from individual mice werecytocentrifuged onto glass slides, air dried and stained withhaematoxylin and eosin for differential cell counts. Lungs were removedfrom mock- and VV-infected mice and single cell suspensions wereprepared by sieving through a 100-μm nylon mesh followed by hypotoniclysis of erythrocytes. Cell viability in all samples was assessed usingtrypan blue exclusion.

BAL cells were counted to determine the numbers of (A) total cells, (B)macrophages and (C) lymphocytes from mock-infected and VV-infected mice.The results are shown in FIG. 4 in which columns represent the mean cellyield per mouse±SEM from groups of 4-5 mice. Columns marked with anasterisk represent mean cell numbers recovered from vΔA44L-infected micethat were significantly different (*, P<0.05) to those of both vA44L andvA44L-rev-infected mice.

Only few cells were recovered from BAL of mock-infected animals 1 dayp.i. with VV, indicating there were only low levels of recruitment tothe lung in the early phase of VV infection (FIG. 4A). BAL cell numbersincreased over time with peak numbers 12 days p.i. for vA44L orvA44L-rev, and 7 days after infection for vΔA44L. Significantly morecells were recovered from vΔA44L-infected mice at day 7 p.i. compared toother VV-infected groups.

Cytospins of BAL cells were prepared and stained with haematoxylin andeosin for differential cell counts and the results are shown in FIGS. 4Band 4C. Uninfected mouse BAL cells were composed almost entirely ofmacrophages, with some (<10%) lymphocytes noted. The majority ofinflammatory cells recruited to the lung during the course of VVinfection were macrophages (FIG. 4B) and lymphocytes (FIG. 4C), withgranulocytes representing less than 5% of total cells at all time pointstested. By day 12 after vΔA44L infection the levels of total cells,macrophages and lymphocytes in BALs were all lower than on day 7,whereas with vA44L and vA44L-rev the cell numbers were higher on day 12than day 7.

Flow Cytometric Analysis of Cell Surface and Intracellular Antigens

Lymphocytes from BAL of VV-infected mice were analysed. Groups of 5BALB/c mice were mock-infected or infected with 10⁴ PFU of vA44L,vΔA44L, or A44L-rev. At 7 days p.i., BAL cells were recovered, andblocked with 10% normal rat serum and 0.5 μg of Fc block (Pharmingen) inFACs buffer (PBS containing 0.1% bovine serum albumin and 0.1% sodiumazide) on ice for 20 min. Cells were stained with fluoresceinisothiocynated (FITC) anti-CD4, Tri-colour anti-CD8 and phycoerythrin(PE)-labelled anti-CD3 or isotype antibody controls (all from Caltag,Burlingame, Calif.). Lymphocytes were identified by their characteristicFSC/SSC profile and by expression of CD3. The distribution of cellsurface markers was determined on a FACScan flow cytometer withCellQUEST software (Becton Dickenson, Mountain View, Calif.). Alymphocyte gate was used to select at least 20,000 events. Data from theexperiments are shown in FIG. 5. The data shown are the mean percentageof BAL cells±SEM from 4-5 individual mice, and are representative of twoindependent experiments.

The results of the flow cytometric analysis at day 7 p.i. is shown inFIG. 5. A greater percentage of both CD4⁺ and CD8⁺ lymphocytes wasobserved in BAL from vΔA44L-infected mice compared to animals infectedwith vA44L or vA44L-rev. Less than 5% of BAL cells were DX5+naturalkiller (NK) cells or B220⁺ B lymphocytes at this time. The data showthat by day 7 p.i. with vΔA44L more cells were recruited to the lungs(FIG. 4) and a higher proportion of those were CD4⁺ and CD8⁺ T cells.

To detect intracellular cytokines, 10⁶ lung cells/ml were stimulatedwith 50 ng/ml PMA (Sigma), 500 ng/ml ionomycin (Calbiochem) in thepresence of 10 μg/ml brefeldin A (Sigma) for 5 h at 37° C. Cells werewashed with FACs buffer and stained with Tri-colour anti-CD4 and FITCanti-CDB for 30 min on ice and then fixed for 30 min at room temperaturewith 2% paraformaldehyde in PBS. Samples were permeabilized with 0.5%saponin in FACS buffer for 10 min. PE-conjugated anti-mouse IFN-γ (cloneXMG1.2 Pharmingen) was added for a further 30 min at room temperatureand the cells were washed once with 0.5% saponin in FACs buffer andtwice in FACs buffer alone. Cells were analyzed on a Becton Dickinsonflow cytometer collecting data on at least 20,000 lymphocytes.Intracellular staining of lung lymphocytes for IFN-γ on days 3, 7 and 10p.i. showed that the proportion of both CD4⁺ and CD8⁺ lymphocytespositive for IFN-γ was higher at days 7 and 10 p.i. in the lungs of miceinfected with VV lacking A44L.

ELISA for Cytokines

Groups of 4-6 BALB/c mice were mock-infected or infected with 10⁴ PFU ofvA44L, vΔA44L, or vA44L-rev. At day 3, 7 and 10 mice were sacrificed,BALs were performed and single cell suspensions were prepared from lungtissue. BAL samples were centrifuged and the levels of IFN-γ present inthe supernatant was determined by ELISA.

IFN-γ, IL-10 and IL-4 in BAL fluid and culture supernatants wasquantified using OptEIA kits from Pharmingen according to theinstructions provided. The concentration of cytokine in each sample wascalculated from a standard curve and expressed as ng/ml. Levels of IFN-γin BAL fluids of VV-infected mice are shown in FIG. 6A. Values representthe mean, with SEM, from 2 groups (n=3/group). The dashed linerepresents the detection limit of the IFN-γ ELISA (50 pg/ml).

As seen in FIG. 6A, IFN-γ levels in BAL fluids from mock-infectedanimals were below the detection limit of the assay. Low levels of IFN-γwere detected in lavage fluids 3 days p.i. and these increased markedlyat days 7 and 10 p.i. Significantly higher levels of IFN-γ were detectedin lavage fluids from vΔA44L-infected mice compared to animals infectedwith control viruses at day 7 p.i. IFN-γ levels at day 10 were alsogenerally higher in mice infected with vΔA44L (FIG. 6A). Levels of IL-10or IL-4 in BAL fluid were below the detection limit of their respectiveELISA assays at each time point tested.

IFN-γ production was determined after culturing lung cell suspensions inthe presence of PMA and ionomycin for 5 h at 37° C. Cells were pelletedand levels of IFN-γ present in the supernatant determined by ELISA. Thedata are shown in FIG. 6B. Values represent the mean±SEM, from twogroups (n=3/group). The dashed line represents the detection limit ofthe IFN-γ ELISA (50 pg/ml). In the Figure, it is seen that IFN-γ levelswere very low in lung cell supernatants from mock-infected mice. Amodest increase was observed in supernatants from lung cells 3 daysp.i., and levels were elevated further from lung cells harvested day 7and day 10 p.i. Consistent with IFN-γ levels observed in BAL,PMA-stimulated lung cells from vΔA44L-infected mice producedsignificantly more IFN-γ than those from animals infected with vA44L orvA44L-rev on day 7 and 10 p.i. (FIG. 6B).

The elevated IFN-γ production detected in BAL fluids and lung cellsuspensions from vΔA44L-infected mice suggested that the additionallymphocytes recruited to the lungs of these animals were producingIFN-γ. Intracellular staining of lung lymphocytes for IFN-γ on days 3, 7and 10 p.i. demonstrated that the proportion of both CD4⁺ and CD8+lymphocytes positive for IFN-γ was higher in the lungs ofvΔA44L-infected mice at days 7 and 10 p.i. (FIG. 6C, D). Only a very lowproportion of CD4⁺ or CD8⁺ cells (<S %) stained for IL-4 or IL-10 in anyof the groups.

To assess intracellular production of IFN-γ by lung lymphocytes frommice 7 days after intranasal VV, lung cells were stimulated with PMA andionomycin for 4 h, brefeldin A was added to retain cytokines in thecytoplasm. Cells were stained with FITC-labelled anti-CD8, APC-labelledanti-CD4, and after permeabilization using saponin, with PE-labelledanti-IFN-γ before analysis by three-color flow cytometry The data areshown in FIGS. 6C and 6D indicating the percentages of CD8⁺ (C) or CD4⁺(D) T cells producing IFN-γ. Values are averaged from two groups(n=3/group). The frequency of IL-4-producing cells was below thedetection limit (<2%) and is not shown. *, P<0.05, **, P<0.02.

Cytotoxic Assays

NK cell cytotoxicity and virus-specific CTL activity were analysed ineffector cells from lung cell suspensions of VV-infected mice. Groups of6 mice were infected with 10⁴ PFU of VV. At days 7 and 10 mice weresacrificed and lung cell suspensions were prepared.

NK cell cytotoxicity and virus-specific CTL activity in lung cellsuspensions was assayed in a standard ⁵¹Cr-release assays. NK-mediatedlysis was tested on YAC-1 cells while P815 cells (H-2^(d), mastocytoma)were used as targets for virus-specific CTL lysis.

Prior to labelling with Na₂ ⁵¹CrO₄ (150 μCi per 3×10⁶ cells) P815 cellswere mock-infected or infected with VV WR at 10 PFU/cell for 2 h at 37°C. Uninfected YAC-1 cells were labelled as above. Serial dilutions ofeffector cells were incubated in triplicate cultures with eitheruninfected or VV-infected target cells in 100 μl of RPMI 1640supplemented with 10% FBS in 96-well V-bottomed plates at 37° C. in 5%CO₂. After 4 h (YAC-1) or 6 h (P815) cells were pelleted and 50 μl ofthe supernatant was transferred to a Lumaplate-96 (Packard InstrumentCompany Inc., USA) and counted using a Packard Microplate Scintillationcounter. The percentage of specific ⁵¹Cr release was calculated as: %specific lysis=[(experimental release−spontaneous release)]/(totaldetergent release−spontaneous release)]×100%. The results of the assaysare shown in FIG. 7. FIGS. 7A and C show data from mice sacrificed after7 days, FIGS. 7B and D show data from mice sacrificed after 10 days.Data are shown for mice infected with vA44L (▪), vΔA44L (◯), orvA44L-rev (▴). Data are expressed as the mean percent specific lysis+SEMfrom two groups of 3 mice plotted against the lung cell:target ratio (A,B) or the CD8⁺ lung cell:target ratio (C, D). Lysis of uninfected P815cells by day 7 and day 10 effector cell populations was always <10% atan effector:target ratio of 100:1.

In some experiments CD8⁺ cells were depleted from lung cell suspensionsby incubation at 37° C. with an anti-CD8 mAb (clone 3.115 (Sarmiento etal., 1980)) in the presence of human complement. Analysis by flowcytometry demonstrated selective depletion of the CD8⁺ cell population.Depleted cells were added to cytotoxicity assays without adjustment forthe depletion in cell number.

At day 5, primary CTL activity was very low in lung cell suspensionsfrom all infected animals (<10% of specific cell lysis), however by day7 significant CTL activity was detected against virus-infected targets(FIG. 7A). The cytolytic activity of lung lymphocytes fromvΔA44L-infected mice was greater than from mice infected with vA44L orvA44L-rev, and this trend was also observed using lung cells fromanimals 10 days p.i. (FIG. 7B). All lung cell suspensions showed veryweak cytotoxic activity against uninfected P815 cells (<10% of specificcell lysis at effector:target ratio of 100:1). Furthermore, nosignificant cytotoxic activity was observed against the NK-sensitiveYAC-1 cell line.

The increase in cytotoxic activity of lung cells from vΔA44L-infectedmice may represent increased recruitment of CTL effectors to the lung oran increased activation state of the lung cells present. To confirm thatCD8⁺ T lymphocytes were mediating CTL activity in our system we treatedday 7 lung cells from WV-infected mice with complement plus a mAb tomurine CD8. This treatment abrogated virtually all CTL activity(undepleted=56% specific lysis, complement alone=46%, complement plusanti-CD8=3%, at effector:target ratio of 100:1).

The numbers of CD8⁺ lymphocytes in the lung cell suspensions weredetermined by flow cytometry, and used to compare CTL activity betweenlung cells based on a CD8⁺ lung cell:target ratio (FIG. 7C, D). Theenhanced lysis observed using lung cells from vΔA44L-infected mice ispartly explained by the higher relative numbers of CD8⁺ T cells in thiscompartment (7.4±2.2%, 13.6±2.0% and 8.0±1.9% of total lung cells at day7, and 14.5±3.8%, 25.1±4.7% and 13.5±3.0% of total lung cells at day 10from mice infected with vA44L, vΔA44L and vA44L-rev, respectively).However, CTL from vΔA44L-infected mice also show an enhanced level oflysis on a per cell basis consistent with an enhanced activation stateof virus-specific CTL within the T cell population (FIG. 7C, D).

Corticosterone Levels in Plasma and Lung

Corticosterone levels in plasma and lungs were measured after intranasalinfection with VV. Plasma and lung extracts were collected from BALB/cmice under low stress conditions after intranasal infection with 10⁵ PFUof vA44L, vΔA44L, or A44L-rev. To guard against fluctuations due tocircadian rhythm, samples were obtained between 9.00 and 10.00 am eachday of assessment. Mice were sacrificed by cervical dislocation andexsanguinated within 4 min of disturbance. Blood was collected inEDTA-coated tubes on ice and was then centrifuged at 3,000 rpm for 10min. Plasma was collected and stored at −20° C. Lungs were removedimmediately after exsanguination, washed once in PBS and placed on ice.Tissue was homogenised and extracted in 2 ml of methanol. Corticosteronelevels in plasma and lung extracts were determined by radioimmunoassayusing a rat corticosterone ³H kit (ICN Pharmaceuticals, Orangeburg,N.Y.). For lung extracts, excess methanol was evaporated and the driedpellet was resuspended in 0.5 ml of the steroid buffer supplied with thekit. Corticosterone levels were determined from individual mice using astandard curve and expressed as ng/ml for plasma or ng/g of lung tissuefor lung extracts.

The results from the assays are shown in FIG. 8. Data represent mean±SEMof 4 or 5 mice per time point and are expressed as ng/ml of plasma or asng/g of lung tissue.

Columns marked with an asterisk represent corticosterone levels fromvΔA44L-infected mice that were significantly different to those fromvA44L- and vA44L-rev-infected mice. *, P<0.05, **, P<0.02.

Titres of infectious virus in the lungs of mice after infection with losPFU of VV were also determined. Virus titres were determined by plaqueassay on BS-C-1 cells and are expressed as PFU/g of lung tissue.Infection with VV induced an increase in corticosterone levels in plasma(FIG. 8A) and lung extracts (FIG. 8B) as early as 1 day p.i., and thelevels increased further up to day 4. Levels were similar in vA44L andvA44L-rev-infected mice at all time points, but were significantly lowerin plasma at day 1 and 2 and in lung extracts at day 2 and 4 fromvΔA44L-infected mice, relative to control-infected animals.Corticosterone is released as part of the acute phase response toinfection and other inflammatory trauma, however viral titres in thelungs were not significantly different between vA44L-, vΔA44L- orvA44L-rev-infected mice at days 1, 2 and 4 p.i. (FIG. 8C), nor werethere differences in visible signs of illness or weight loss at theseearly time points at this dose or at 10⁴ PFU (FIG. 2). The enhancedlevels of endogenous GCs observed in vA44L and vA44L-rev-infected micesuggest that A44L may have a direct effect upon local and systemicsteroid levels during VV infection.

VV Expressing a Gene or Gene Fragment Encoding a Foreign Antigen butLacking A44L

The gene encoding the antigen is introduced into the VV in a similarmanner to that described above in relation to deletion of A44L. Cellsare infected with VV and transfected with a plasmid recombinationvector. The is plasmid contains a gene that encodes the antigen. Theplasmid also comprises a correctly oriented VV promoter, a W earlytranscriptional termination sequence (TTTTTNT) flanked by two DNAsequences homologous to sequences of the VV that may be disruptedwithout compromising the viability of the VV.

Within the infected cells, homologous recombination between the VVgenome and the plasmid DNA results in insertion of the foreign gene intothe VV genome. The recombinant genome is replicated and packaged intoinfectious progeny virus. The progeny virus is screened for desiredrecombinants and insertion of the desired gene is confirmed by PCR.

VV Vaccine

10⁶ to 10⁷ infectious particles of the recombinant VV are freeze driedin 100 μl of PBS in the presence of 2% peptone and 1% human albumin inan ampoule, preferably a glass ampoule. The ampoule is then sealed andstored at a temperature below −20° C.

For vaccination, the lyophilisate is made up to 0.1 ml of aqueoussolution with physiological saline. The vaccine is administered byintradermal inoculation.

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All publications and patent applications cited in this specification areincorporated herein by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A method of vaccinating a subject comprising administering to thesubject an immunogenic agent, wherein the immunogenic agent is arecombinant poxvirus, wherein the recombinant poxvirus genome does notcomprise a functional gene encoding a 3β-hydroxysteroiddehydrogenase/Δ⁵-Δ⁴isomerase.
 2. (canceled)
 3. (canceled)
 4. (canceled)5. (canceled)
 6. (canceled)
 7. (canceled)
 8. (canceled)
 9. (canceled)10. (canceled)
 11. (canceled)
 12. (canceled)
 13. (canceled)
 14. Themethod of claim 1 wherein the recombinant poxvirus is selected from thegroup consisting of orthopoxviruses, parapoxviruses, avipoxviruses,suipoxviruses, molluscipoxviruses, and yatapoxviruses.
 15. (canceled)16. (canceled)
 17. (canceled)
 18. (canceled)
 19. A The method of claim1, wherein the recombinant poxvirus has no coding sequence encoding a3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴ isomerase; or wherein the geneencoding the 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴isomerase isdisrupted, mutated or truncated such that its gene product has reducedactivity; or wherein one or more mutations or deletions in the promoteror other upstream sequences of the gene encoding a 3β-hydroxysteroiddehydrogenase/Δ⁵-Δ⁴isomerase cause expression of the gene to becompromised, leading to reduced levels of gene expression. 20.(canceled)
 21. (canceled)
 22. (canceled)
 23. A vaccine compositioncomprising a recombinant poxvirus wherein the poxvirus genome does notcomprise a functional 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴isomerase,and a pharmaceutically suitable carrier.
 24. The vaccine compositionaccording to claim 23, wherein said composition further comprises one ormore additives selected from the group consisting of a preservative, astabiliser and an adjuvant.
 25. The vaccine composition according toclaim 23, wherein said recombinant poxvirus is selected from the groupconsisting of vaccinia viruses, parapoxviruses, avipoxviruses,suipoxviruses, molluscipoxviruses, and yatapoxviruses.
 26. (canceled)27. A vaccine composition according to claim 23, wherein the recombinantpoxvirus has no coding sequence encoding a 3β-hydroxysteroiddehydrogenase/Δ⁵-Δ⁴isomerase; or wherein the gene encoding the3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴isomerase is disrupted, mutated ortruncated such that its gene product has reduced activity or wherein oneor more mutations or deletions in the promoter or other upstreamsequences of the gene encoding a 3β-hydroxysteroiddehydrogenase/Δ⁵-Δ⁴isomerase cause expression of the gene to becompromised, leading to reduced levels of gene expression.
 28. Themethod of claim 1, wherein said immunogenic agent is a recombinantpoxvirus having a genome comprising a non-poxvirus gene or a fragment ofa non-poxvirus gene which gene or fragment encodes an antigen. 29.(canceled)
 30. The method of claim 28, wherein the poxvirus is avaccinia virus, a cowpox virus, a camelpox virus or an ectromelia virus,or a derivative of any of those viruses.
 31. (canceled)
 32. The methodof claim 28, wherein the poxvirus is a vaccinia virus strain selectedfrom the group consisting of Lister, Copenhagen, Wyeth, New York CityBoard of Health, NYVAC, Praha virus, DRYVAX Wyeth-derived virus, LIVP,IHD-J, IHD-W, Tian Tan, Tashkent, King Institute, Patwadanger, EM-63,Evans, Bern, LC16m0 and MVA.
 33. (canceled)
 34. The method of claim 28,wherein the non-poxvirus gene or non-poxvirus gene fragment that encodesan antigen is a non-poxvirus gene or non-poxvirus gene fragment againstthe gene product of which a protective immune response in a subject isdesirable.
 35. (canceled)
 36. (canceled)
 37. The method according toclaim 28, wherein the administration of said immunogenic agent is forthe prophylaxis of an infection caused by a pathogenic agent, or for theprophylaxis or treatment of a disease associated with aberrant cells.38. The method according to claim 37, in which the non-poxvirus geneencodes an immunogenic peptide or polypeptide of an infectious pathogen,or an antigenic peptide or polypeptide of aberrant cells, theelimination or induced quiescence of which is beneficial.
 39. The methodaccording to claim 28, wherein the recombinant poxvirus is selected fromthe group consisting of parapoxviruses, avipoxviruses, suipoxviruses,molluscipoxviruses and yatapoxviruses.
 40. The method according to claim28, wherein the non-poxvirus gene that encodes an antigen is anon-poxvirus gene against the gene product of which a protective immuneresponse in a subject is desirable.
 41. The method according to claim28, wherein the poxvirus has no coding sequence encoding a3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴isomerase, or wherein the geneencoding the 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴isomerase isdisrupted, mutated or truncated such that its gene product has reducedactivity; or wherein one or more mutations or deletions in the promoteror other upstream sequences of the gene encoding a 3β-hydroxysteroiddehydrogenase/Δ⁵-Δ⁴isomerase cause expression of the gene to becompromised, leading to reduced levels of gene expression. 42.(canceled)
 43. (canceled)
 44. A vaccine composition comprising apoxvirus having a genome comprising a non-poxvirus gene or a fragment ofa non-poxvirus gene which gene or fragment encodes an antigen, whereinthe poxvirus genome does not comprise a functional gene encoding a3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴isomerase, and a pharmaceuticallysuitable carrier.
 45. The vaccine composition according to claim 44,further comprising one or more additives selected from the groupconsisting of an antibiotic, a preservative, a stabiliser and anadjuvant.
 46. The vaccine composition according to claim 44, whereinsaid poxvirus is selected from the group consisting of a vaccinia virus,a cowpox virus, a camelpox virus, and an ectromelia virus, andderivatives thereof.
 47. (canceled)
 48. (canceled)
 49. (canceled)
 50. Arecombinant poxvirus having a genome comprising a non-poxvirus gene or afragment of a non-poxvirus gene which gene or fragment encodes anantigen, wherein the poxvirus genome does not comprise a functional geneencoding a 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴isomerase, with theproviso that the non-poxvirus gene or fragment of a non-poxvirus gene isnot a gene encoding varicella-zoster virus glycoprotein E, hepatitis Bvirus preS2-S protein or E. coli guanine phosphoribosyl transferase 51.The vaccine composition of claim 44, wherein said recombinant poxvirusis a vaccinia virus strain selected from the group consisting of Lister,Copenhagen, Wyeth, New York City Board of Health, NYVAC, Praha virus,DRYVAX Wyeth-derived virus, LIVP, IHD-J, IHD-W, Tian Tan, Tashkent, KingInstitute, Patwadanger, EM-63, Evans, Bern, LC16m0 or MVA.
 52. Thevaccine composition of claim 44, wherein said poxvirus is selected fromthe group consisting of parapoxviruses, avipoxviruses, suipoxviruses,molluscipoxviruses, and yatapoxviruses.
 53. The vaccine composition ofclaim 44, wherein the recombinant poxvirus has no coding sequenceencoding a 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴isomerase; or whereinthe gene encoding the 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴isomerase isdisrupted, mutated or truncated such that its gene product has reducedactivity; or wherein one or more mutations or deletions in the promoteror other upstream sequences of the gene encoding a 3β-hydroxysteroiddehydrogenase/Δ⁵-Δ⁴isomerase cause expression of the gene to becompromised, leading to reduced levels of gene expression.